One of the key pieces missing in understanding the regulation of hemostasis is the mechanism by which the vitamin K-dependent (VKD) clotting factors are carboxylated. Carboxylation is a post-translational modification that proteins like prothrombin, factor IX and factor VII require for activity. It involves the conversion of clusters of glutamyl residues to gamma carboxyglutamyl residues in a reaction that requires vitamin K hydroquinone and is inhibited by the anticoagulant Warfarin, a vitamin K antagonist. Carboxylase activity resides in an integral membrane protein present in most tissues. Carboxylated VKD proteins have also been isolated from bone and smooth muscle. Whether there are multiple carboxylases for the different VKD proteins or only a single enzyme remains an open question. The carboxylation of VKD proteins is poorly understood. Most of the studies have been performed using crude microsomal preparations on small synthetic peptide analogs based on sequences derived from the VKD proteins. The carboxylase cDNA has been isolated, but the protein encoded by it does not share homology with other known proteins, and the cDNA provides little suggestive information about carboxylase structure or function. Dr. Berkner's lab has developed a novel, model system for analyzing the intracellular interaction of the carboxylase with individual recombinant VKD proteins in mammalian cells. This system allows for isolation of the carboxylase-VKD protein enzyme-substrate complex for in vitro analysis. Dr. Berkner has also constructed cell lines that over-express the carboxylase and can now isolate large amounts of pure, active human carboxylase. Long-term goals are to understand the mechanism of carboxylation and the role that the carboxylase plays in multiple biological systems. Specific aims for this grant period are to: 1) analyze the domain organization of the carboxylase; 2) analyze the mechanism of VKD protein carboxylation by identifying the limiting step in the secretion of carboxylated proteins; 3) test whether there are multiple carboxylases or if one carboxylase modifies all VKD proteins. Data from these experiments will elucidate why the carboxylation of VKD proteins is limited in vivo even when the carboxylase is overexpressed and how the carboxylase carries out this unusual, and critical, reaction. These experiments will also provide methods for the future isolation of VKD proteins and information for the design of anticoagulants that specifically alter hemostasis.